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系統識別號 U0026-0812200915233603
論文名稱(中文) 探討C型肝炎病毒之五端非轉譯區與非結構蛋白 2 和 3 的共同演化關係
論文名稱(英文) Co-evolution of the 5'-untranslated region, nonstructural proteins 2 and 3 of hepatitis C virus
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系碩博士班
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 97
學期 2
出版年 98
研究生(中文) 歐乃瑛
研究生(英文) Nai-Ying Ou
電子信箱 milking1013@hotmail.com
學號 T3696107
學位類別 碩士
語文別 英文
論文頁數 98頁
口試委員 口試委員-賴明德
口試委員-張定宗
口試委員-鄭如茜
口試委員-王憲威
指導教授-楊孔嘉
中文關鍵字 五端非轉譯區  共同演化  次基因複製子  非結構蛋白2  非結構蛋白3  C型肝炎病毒  生物資訊 
英文關鍵字 HCV  5' UTR  co-evolution  data mining  subgenomic replicon  NS3  NS2 
學科別分類
中文摘要 C型肝炎病毒 (HCV) 感染是造成慢性肝炎、肝硬化以及肝癌的主因之一,由於C型肝炎的非結構蛋白5B (NS5B) 所轉譯的以RNA為模板進行複製的RNA聚合酶 (RdRp) 缺乏了校正的功能,再加上C型肝炎病毒具有很高的複製能力,在快速複製的過程中因為無法校正而造成C型肝炎病毒具有很高的基因歧異度。因此在此研究中,我們從 Los Alomas HCV資料庫中下載了217條由病人身上取得的全長C型肝炎病毒序列,並利用生物資訊軟體 Weka 找出在不分基因型的情況下,C型肝炎病毒五端非轉譯區 (5'UTR),非結構蛋白2 (NS2),以及非結構帶蛋白3 (NS3) 共有9組共同演化配對 (Co-evolutionary pair),為了探討這些共同演化配對對於C型肝炎病毒複製能力的影響,我們利用HCV次基因複製子 (Subgenomic replicon) 為工具,研究位於5'UTR核甘酸第243位點由鳥糞嘌呤 (Guanine) 轉變為腺嘌呤 (Adenine) 分別配對NS2胺基酸第14位點由苯丙胺酸 (Phenylalanine) 轉變為白胺酸 (Leucine)、第41位點由異白胺酸 (Isoleucine) 變為白胺酸 (Leucine)、第76位點由異白胺酸 (Isoleucine) 變為纈氨酸 (Valine)、第110位點由異白胺酸 (Isoleucine) 變為白胺酸 (Leucine)、第211位點由甘胺酸 (Glycine) 變為絲胺酸 (Serine)、第212位點由麩醯胺酸 (Glutamine) 變為離胺酸 (Lysine),以及 5'UTR核甘酸第243位點由鳥嘌呤 (Guanine)轉變為腺嘌呤 (Adenine) 分別配對NS3胺基酸第71位點由異白胺酸 (Isoleucine) 變為纈氨酸 (Valine)、第175位點由甲硫胺酸 (Methionine) 變為白胺酸 (Leucine)、第621 位點由丙胺酸 (Alanine) 變為蘇胺酸 (Threonine) 的交互影響。實驗結果顯示,NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S, NS3-I71V 以及NS3-M175L單一位置突變會降低HCV複製能力,但若這些突變與5'UTR-G243A同時存在,則會回復HCV複製能力。但在NS2-F14L、NS2-Q212K以及NS3-A621T則無這種補償現象存在。除此之外,當NS2-I76V以及NS3-I71V兩個突變同時存在時,C型肝炎病毒的複製能力也會下降,但若此兩個突變與5'UTR-G243A同時存在,C型肝炎病毒複製能力也能被補償回來。接著,在NS2蛋白不存在的情況下,NS3-I71V及NS3-M175L的存在會使得HCV複製能力下降,且此下降的情形無法經由5'UTR的存在而補償,這樣的結果代表著NS2蛋白也參與在5'UTR核甘酸第243位點以及NS3蛋白胺基酸第71以及175位置共同演化的過程中。接著,我們將5'UTR由鳥嘌呤 (Guanine)轉變為腺嘌呤 (Adenine)、胞嘧啶 (Cytosine)、胸腺嘧啶 (Thymidine) 後,我們發現在2-3'subgenomic replicon 中,5'UTR-G243C的突變會造成subgenomic replicon的複製能力下降約一半左右,而5'UTR-G243T的突變則使得HCV複製能力幾乎完全消失,但在3-3'subgenomic replicon 中,5'UTR-G243A, T, C的突變對於subgenomic replicon的複製能力則幾乎沒有任何影響,因此我們認為5'UTR第243位點的突變會影響5'UTR以及NS2蛋白之間的作用而造成複製能力下降。因此接下來我們利用RNA免疫沉澱法來探討5'UTR第243位點以及NS2蛋白之間的交互作用。而結果顯示帶有NS2-F14L,I41L,I76V,I110L,G211S以及Q212K突變的NS2蛋白會和野生型的3-3'subgenomuc replicon有交互作用,而野生型的NS2蛋白則否。而3-3'-5'UTR-G243A subgenomuc replicon 和野生型 NS2蛋白、NS2-F14L之間的交互作用比3-3'-5'UTR-G243A subgenomuc replicon 和NS2-I41L,I76V,I110L,G211S以及Q212K突變的NS2蛋白來的強烈,結果也顯示當HCV RNA和NS2蛋白的交互作用越強烈,HCV subgenomuc replicon的複製能力就越差,代表這些共同演化配對的交互作用會影響HCV的複製。綜合以上結果,我們的實驗發現位於NS2,NS3的特定胺基酸會和5'UTR的核甘酸共同演化而保有HCV複製的能力。
英文摘要 Hepatitis C virus (HCV) has been recognized as one of the major causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC). The nonstructural (NS) protein 5B of HCV is a RNA-dependent RNA polymerase (RdRp) which lacks a proof-reading function, together with the high replicative activity, is the basis of high genetic variability of HCV. In this study, 217 full-length HCV sequences were downloaded from Los Alomas HCV database, analyzed with the use of data mining software Weka, and we found that there existed 9 intra-genotypic co-evolutionary pairs between NS2, NS3 and 5'UTR in naturally occurring HCV strains. In this study, co-evolutionary pairs of the 243rd nucleotide of 5'-untranslated region (5'UTR) and the 14th, 41st, 76th, 110th, 211th, 212th amino acids of NS2 as well as the 71st, 175th, 621st amino acids of NS3 were examined to find out if these co-evolutionary mutations will regulate HCV replication. The HCV subgenomic replicon carrying single or multiple mutations were electroporated into cured-Huh7 cells and luciferase reporter activities were analyzed. The results showed that NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S, NS3-I71V and NS3-M175L single mutation down-regulated HCV replication when compared with the wild-type HCV replicon, but as these mutations were combined with 5'UTR ntG243A, the replicative activities could be compensated. The NS2-F14L, NS2-Q212K, and NS3-A621T single mutation also attenuated HCV replicative activities and these impairment effects could not be compensated as combined with 5'UTR ntG243A. The cell-based assay thus supported that the 14th, 41st, 76th, 110th, 211th of NS2, and 71st, 175th of NS3 might modulate HCV replication in concert of 5'UTR nt243. Furthermore, the combination of NS2-I76V and NS3-I71V also has compensatory effect with 5'UTR-G243A. In the absence of NS2 protein, the NS3-I71V and NS3-M175L mutations abolished HCV replication. These results indicated that NS2 protein play a role in modulating the co-evolution between 5'UTR-nt243 and 71st, 175th amino acids of NS3. Besides, the 5'UTR-G243C downregulated HCV replication activities to about half and the substitution of 5'UTR-G243T almost eliminated the HCV replication activities in 2-3'sibgenomic replicon, but the substitutions of 5'UTR-G243A, T, C had no influences on 3-3'subgenomic replicon, indicating that the substitution of 5'UTR nt243 might interfere the interaction between 5'UTR and NS2 protein thus modulating HCV replication activities. Finally, RNA-immunoprecipitation assay was used to investigate whether there are direct interactions between 5'UTR-nt243 and NS2 wt and mutated proteins. The results indicated that there were interactions of HCV genome with NS2 mutated proteins (NS2-F14L, NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S, NS2-Q212K) but not NS2-wt proteins in Feo-3-3' wild-type stable cells. In Feo-3-3'-G243A stable cells, however, interactions of HCV genome with NS2-wt, NS2-F14L were much stronger than those with NS2-I41L, NS2-I76V, NS2-I110L, NS2-G211S and the stronger the interactions between HCV RNAs and NS2 mutated proteins, the lower the replication of HCV, suggesting that the interactions between the co-evolutionary pairs might impair HCV replication. In conclusion, amino acid residues at NS2 and NS3 might co-evolve with nucleotide of 5'UTR-nt243, thus entails reservation of HCV replication.
論文目次 Abstract (in Chinese)....................................I
Abstract (in English)..................................III
Acknowledgements.........................................V
Index...................................................VI
Tables/Figures...........................................X
Reagents and Instruments...............................XII
Chapter 1: Introduction..................................1
1. Hepatitis C virus (HCV)...............................2
1.1 The epidemiology of HCV..............................2
1.2 HCV genotypes........................................3
1.3 HCV database.........................................4
1.4 The molecular virology of HCV........................5
1.5 The characteristic of 5’UTR, NS2 and NS3 of HCV....10
1.6 RNA-protein interaction in HCV......................13
2. Bioinformatic research...............................14
2.1 Data-mining and Association rule..................14
2.2 Weka................................................15
3. Study goal and experimental strategy.................16

Chapter 2: Materials and Methods........................18
1. Bioinformatic research for co-evolutionary sites.....20
1.1 Sequences collection................................20
1.2 Phylogenetic tree analysis..........................20
1.3 Sequence components for data mining in
bioinformatics......................................20
1.4 Data-mining analysis................................21
2. Cell line and culture system.........................21
2.1 Cured-Huh7 cell line................................21
2.2 Cell culture system.................................21
2.3 Preparation of frozen cells.........................23
2.4 Defrozen cells......................................24
3. Preparation of mutated replicons for studying of
co-evolution.........................................24
3.1 Site-directed mutagenesis by QuickChange XL Site directed mutagenesis kit................................24

3.2 Transform of Dpn1-treated DNA.......................27
3.3 Mini-plasmid extraction.............................27
3.4 Midi-plasmid extraction.............................29
3.5 Restriction enzyme digestion........................30
3.6 Gel extraction......................................31
3.7 Ligation............................................32
3.8 Replicon linearization..............................33
3.9 In vitro transcription..............................33
3.10 RNA purification...................................34
3.11 Total RNA extraction from cured-Huh7 cells.........36
3.12 Electroporation....................................37
3.13 Luciferase activity assay..........................38

4. Interaction between HCV RNA and NS2 protein..........39
4.1 Construction of NS2, NS3 expressing proteins........39
4.2 Transfection........................................40
4.3 Protein concentration detection.....................41
4.4 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)..............................41
4.5 Protein transfer and blotting.......................43
4.6 Immunoprecipitation of RNA-protein complexes........45

Chapter 3: Results......................................48
1. Phylogenetic tree of 217 HCV sequences...............50
2. Co-evolutionary pairs predicted between the nucleotide sequences of HCV........................................50
UTRs and the amino acid sequences of NS2, NS3, NS5B by association rules
3. The co-evolutionary pairs of HCV 5’UTR and NS2 proteins, HCV 5’UTR and NS3 proteins have compensatory effects on HCV replication..............................50
4. Different codon usages of NS2-I110L and NS2-G211S to confirm the compensatory effects between 5’UTR and NS2 proteins................................................52
5. The co-evolutionary pair of 5’UTR-G243A, NS2-I76V
and NS3-I71V has compensatory effect on HCV replication.52
6. The substitutions of 5’UTR nt243G, A, T, C have different influences on the replication activities of HCV 2-3'and 3-3'subgenomic replicons........................53
7. The NS3-I71V and NS3-M175L mutations impaired HCV 3-3' subgenomic replicon replication.........................53
8. NS2 wt and mutated proteins can express when transfected into cured-Huh7 cells and the protease inhibitor can increase the expression level of NS2
proteins. ..............................................54
9. The co-evolutionary pairs of HCV 5’UTR and NS2 proteins have compensatory effects on HCV replication (Reverse method).......................................54
10. There are some interaction between HCV 5’UTR nt243 and NS2 wt and mutated proteins........................55
11. The cluster of NS2-I41L-I76V-I110L-G211S mutations decreased the NS2 protein expression...................56

Chapter 4: Discussion..................................57
1. Co-evolution pairs predicted by bioinformatics
tools...............................................58
2. The co-evolutionary effects of 5’UTR nt243 and NS2,
NS3 proteins........................................59
3. The influence of substitutions at 5’UTR nt243......60
4. Impairment of NS3-I71V, NS3-M175L mutations in 3-3’
subgenomic replicons................................61
5. Interactions between 5’UTR-G243A and NS2 proteinn..62
6. Conclusion..........................................63
References.............................................64
Tables/Figures.........................................72
Author.................................................98
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